Gdnf-related neuropeptides

ABSTRACT

The invention provides novel neuropeptides having amino acid sequences derived from GDNF precursors and homologs thereof, as well as compositions containing the novel neuropeptides. The invention also provides polynucleotides encoding the neuropeptides, antibodies that specifically bind to the neuropeptides, and methods of making and using all of the above, particularly in the treatment of motor disorders, neuropathic pain, and for modulating excitatory neurotransmission.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationNo. 60/500,613, filed Sep. 5, 2003, the disclosure of which is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to neuropeptides derived from theprecursor of glial cell line-derived neurotrophic factor (GDNF) andthose of GDNF homologs, and use of these novel neuropeptides.

BACKGROUND OF THE INVENTION

Neuropeptides are polypeptides that serve effector functions in thenervous system. Most vertebrate polypeptide hormones and neuropeptidesof the constitutive secretory pathway are synthesized as largebiologically inactive proproteins and are activated throughendoproteolytic cleavage of the propeptide. The enzymes responsible forthe activation: furin, PACE4, PC4, PC5/6 and PC7/8, are members of theprohormone convertase (PC) family. Known substrates include pro-β-nervegrowth factor (Bresnahan P. et al. (1990) J. Cell Biol. 111:2851-2859),proinsulin-like growth factor-IA (Duguay S. et al. (1997) J. Biol. Chem.272(10): 6663-6670) and bone morphogenic protein-4 (BMP-4) (Cui Y. etal. (1998) EMBO J. 17(16): 4735-4743; Constam D. and E. Robertson (1999)J. Cell Biol. 144(1): 139-149).

GDNF is a protein that is initially synthesized as a larger proprotein(precursor) and is subsequently proteolytically processed to form matureGDNF. GDNF belongs to the transforming growth factor β (TGFβ)-family ofproteins, which are characterized by a “cysteine knot”: a pattern ofintertwined loops consisting of three intramolecular disulfide bonds. Inaddition, one of the cysteines conserved within the family is used forhomodimer formation. These bonds are formed already in the endoplasmicreticulum after translocation and signal sequence removal, whereas furinhas been localized in trans-Golgi (Shapiro J. et al. (1997) J.Histochem. Cytochem. 45(1): 3-12). The cysteine-bonding pattern ofsubstrates, such as GDNF precursor, affects the accessibility of theprotein to processing.

GDNF signals via its receptor cRet, a receptor tyrosine kinase, and theco-receptor GDNF family receptor α1 (GFRα1). Other co-receptors act inconjunction with cRet to transduce a signal for other ligands. Forinstance, cRet and the co-receptor GFRα2 are required for neurturinsignalling, cRet and its co-receptor GFRα3 are required for arteminsignalling, and cRet and GFRα4 are required for persephin signalling.Evolutionary studies indicate that GDNF could represent an ancestralform of neurotrophic factors.

In addition to neuronal tissue, GDNF has been found to be expressed in avariety of non-neuronal tissues of animals including developing skin,whisker pad, kidney, stomach, testis, developing skeletal muscle, ovary,lung, and adrenal gland (Trupp, M. et al. (1995) J. Cell Biol. 130(1):137-48). GDNF has been found to play a role in nephrogenesis (Moore, M.W. et al. (1996) Nature 382(6586):76-9); apoptosis-driven hair follicleinvolution (Botchkareva, N. V. et al. Am. J. Pathol. 156(3):1041-1053);heart development (Hiltunen, J. O. (2000) Dev Dyn 219(1):28-39); andmaintenance of the adult enteric nervous system (Peters, R. J. et al.(1998) J. Auton Nerv. Syst. 70(1-2): 115-22). Both the rodent and humanGDNF precursors contain several putative target sites for the successiveactions of PCs and the enzymes required for C-terminal amidation,carboxypeptidase E (CPE) and peptidylglycine a-amidating monoxygenase(PAM) (FIG. 1 A). Areas and timing of expression of these enzymesoverlap also the sites of GDNF synthesis (Shafer et al. (1993) J.Neurosci. 13(3): 1258-1279, Zheng et al. (1997) Dev. Biol. 181(2):268-283, Zhang et al. (1997) Dev. Biol. 192(2): 375-392). Cleavage ofproGDNF at the PC substrate sites could result in the formation ofseveral amidated peptides (FIG. 1, Table 1).

The polypeptides of the invention have effects in the nervous system. Inaddition, the polypeptides of the invention may have effects outside ofthe nervous system as well. Such activities are also embraced by theinvention. It is known for some neuropeptides that the neuropeptideshave other effects apart from the nervous system. For example, theyregulate the secretory functions of adrenal cortex and pancreas(Renshaw, D. et al (2000) Endocrinol. 141(1): 169-73 and act asimmunomodulators (Krantic S. (2000) Peptides 21(12):1941-64; WiedermannC. J. (2000) Peptides 21(8):1289-98). Therefore, the invention embraceseffects of the neuropeptides acting in the nervous system and apart fromthe nervous system.

Interaction of GDNF with its receptors cRet and GFRα1 has been suggestedto have a role in a variety of diseases and disorders including:neuropathic pain (Boucher, T. J. et al. (2000) Science 290(5489):124-7); motor diseases such as amyotrophic lateral sclerosis (ALS) andX-linked spinal and bulbar muscular atrophy (SBMA) (Yamamoto, M. et al.(1999) Neurochem. Res. 24(6): 785-90); neurodegenerative diseases suchas Parkinson's disease (Walton, K. M. (1999) Mol. Neurobiol. 19(1):43-59); renal diseases (Onodera, H. et al. (1999) Nephrol. Dial.Transplant. 14(6): 1604-5); and epileptic syndromes (Kokaia, Z. et al.(1999) Eur. J. Neurosci. 11(4): 1202-16).

There is a need in the art to further study GDNF, and to developeffective treatments for motor neuropathies, neurodegenerative diseases,renal disorders and neuropathic pain based on the interaction of GDNFand homologs thereof with their receptors.

SUMMARY OF THE INVENTION

The present invention provides isolated neuropeptides derived from theGDNF precursor and its homologs. Surprisingly, polypeptides of theinvention have an effect on hippocampal neurones that mimics or isopposite to the effect of neuropeptide Y (NPY) and polypeptide YY (PYY).The GDNF precursor and homologs may be derived from animal GDNFs,including, but not limited to mammalian and vertebrate GDNFs. Examplesof GDNF precursor and homologs include, but are not limited toprecursors of human GDNF (SEQ ID NO:1), mouse GDNF (SEQ ID NO:2), ratGDNF (SEQ ID NO:3), and chicken GDNF (SEQ ID NO:4). The invention alsoprovides isolated neuropeptides derived from human GDNF precursorcomprising the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:12, and SEQ ID NO:15.

The invention further provides isolated neuropeptides derived from themouse GDNF precursor protein comprising the amino acid sequence of SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:16, as well asisolated neuropeptides derived from the rat GDNF precursor proteincomprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, and SEQ ID NO:16.

The invention also provides isolated neuropeptides derived from thechicken GDNF precursor protein comprising the amino acid sequence of SEQID NO:14 and SEQ ID NO:17.

Alpha-amidated neuropeptides are also provided. In specific embodiments,the alpha-amidated neuropeptides have the amino acid sequence of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15, and SEQ ID NO:16, or SEQID NO:17.

The invention provides polynucleotide sequences encoding the variousneuropeptides of the invention. Plasmids comprising polynucleotidesencoding the isolated neuropeptides of the invention are also embracedby the invention. In certain embodiments, the plasmids are expressionvectors for expressing the polynucleotides encoding the neuropeptides ofthe invention.

The invention also provides transformed cells in which a prokaryotic oreukaryotic cell is transfected with a plasmid comprising thepolynucleotides encoding the neuropeptides of the invention. The cellscan be of vertebrate, invertebrate, bacterial or yeast origin. Inpreferred embodiments, the cells are of vertebrate, specificallymammalian, origin.

The invention provides methods for producing the neuropeptides of theinvention such as through recombinant molecular technology, chemicalsynthesis, and purification from natural sources. The peptides may alsobe cleaved in vitro after first isolating larger precursors from cells.

The invention also provides methods of modulating neuronal responses byadministering to an animal the isolated polypeptides derived from GDNFprecursor. In yet a further aspect, the present invention relates to amethod for modulating neuronal responses in mammals by administering toan animal the isolated polypeptides derived from the GDNF precursor.

The invention also provides methods of modulating responses innon-neuronal tissues both in vivo and in vitro. In preferred embodimentsthe non-neuronal tissues include, but are not limited to renal tissue,testicular tissue, skin, cardiac tissue, gastrointestinal tissue,skeletal muscle, ovarian tissue, lung tissue, and adrenal tissue.

In yet another aspect, the present invention relates to a probe foridentifying GDNF neuropeptide receptors in vertebrates. The probecomprises a polypeptide derived from GDNF or a GDNF homolog. In specificembodiments, the probes have the amino acid sequence of SEQ ID NOs:9-17.

The invention also provides methods of modulating excitatory neuronaltransmission by administering to a subject an effective amount of atleast one neuropeptide of the invention. In some embodiments, theinvention provides methods of modulating excitatory neuronaltransmission by administering to a subject an effective amount of atleast one neuropeptide of the invention.

The invention further provides methods of modulating cRet byadministering compositions that contain at least one neuropeptide of theinvention.

The invention further provides methods of modulating NPY—Y_(i) receptorsby administering compositions that contain at least one neuropeptide ofthe invention.

The invention also provides compositions and methods for treating motordisorders such as ALS; neurodegenerative disorders such as Parkinson'sDisease; epileptic syndromes; renal disorders; skin disorders;testicular disorders; and neuropathic pain. The compositions contain atleast one GDNF precursor derived neuropeptide, homolog, or fusionprotein thereof, or at least a portion of an anti-neuropeptide antibodythat specifically binds to a GDNF precursor derived neuropeptide orhomolog thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawings willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1. An amino acid sequence alignment of the precursors of human GDNF(SEQ ID NO:1), mouse GDNF (SEQ ID NO:2), rat GDNF (SEQ ID NO:3) andchicken GDNF (SEQ ID NO:4). The proregions of each GDNF precursor areshown overlined with an arrow and include: human (SEQ ID NO:5), mouse(SEQ ID NO:6), rat (SEQ ID NO:7) and chicken (SEQ ID NO:8), GDNFproregion. The cleavage sites are C-terminal to the sites marked in boldletters. Cysteine residues are indicated with boxes, and the mature GDNFsequence for human, mouse, rat, and chicken (SEQ ID:s 20-23,respectively) is doubly overlined. Putative prohormone convertase (PC)processing sites in human, mouse, rat and chicken GDNF are shown inboldface letters.

FIG. 2. Amino acid sequence alignment of hPEP2 (SEQ ID NO:10), rPEP2(SEQ ID NO:11), hPYY (SEQ ID NO:19), and hPYY2 (SEQ ID NO:18).

FIG. 3. Binding of iodinated peptides SEQ ID NO:9 and SEQ ID NO:11 torodent tissues. The I¹²⁵-labelled SEQ ID NO:11 displayed intensivebinding to slices of adult rat brain (A-B), in contrast to I¹²⁵-labelledSEQ ID NO:10 (C-D). The bright (A, C) and dark field (B, D) images ofsections containing the hippocampal area from the peptide-incubatedbrain tissue are shown. I¹²⁵-labelled SEQ ID NO:9 (E, H) bound toembryonic (E15) mouse tissues. In the gut (E, F) and the metanephrickidney capsule (G, H), the binding of I¹²⁵-labelled SEQ ID NO:9 (E, G)and the immunohistochemical staining with neuron-specific Tuj1 antibody(F, H) have similar distributions. In testis, I¹²⁵-labelled SEQ ID NO:9binding to the interstitium (I) was complementary both to the expressionof GDNF and to the localization of neurons with Tuj1 within theseminiferous tubules (J).

FIG. 4. FIG. 4. SEQ ID NO:11 (BEP) increases synaptic transmission via aNEM (N-ethylmaleimide)-sensitive receptor in the adult rat hippocampus.Examples of individual recordings of the effect of SEQ ID NO:11 on thestimulus-induced population spikes (pSpikes) and the populationexcitatory postsynaptic potentials (pEPSPs) in the absence (A) or thepresence (B) of NEM are shown. The increase in presynaptic volley isseen only in the absence of NEM (A). Empty arrowheads indicatestimulation. Summaries of the measurements of pSpikes (C) and pEPSPs (D)are shown as fractions of control (control=1). SEQ ID NO:11, but not SEQID NO:9 (NRP) or SEQ ID NO:15 (hPEP4), substantially increases bothpSpikes and pEPSPs. NEM alone also increases both pSpikes and pEPSPs,but no further increase is detected when also SEQ ID NO:11 is added. In(C) and (D) the effect of SEQ ID NO:11 is compared to the baseline atthe beginning of an experiment. 10 nM SEQ ID NO:11, 20 nM SEQ ID NO:9and SEQ ID NO:15, and 250 μM NEM was used. ns=not significant.

FIG. 5. Binding of the control peptide SEQ ID NO:15 to embryonic mousetestis (A) and gut (B).

FIG. 6. Effects of increasing SEQ ID NO:11 concentrations on pEPSP andpSpike. 10 nM SEQ ID NO:11 was chosen for further experiments.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The practice of the present invention, unless otherwise indicated,employs conventional methods of molecular biology and recombinant DNAtechniques known to those of ordinary skill of the art. Such techniquesare known and fully explained in the literature, and are available insuch reference texts as Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL (2nd Edition, 1989); DNA CLONING: A PRACTICALAPPROACH, Vols. I & II (D. Glover, ed.); METHODS IN ENZYMOLOGY (S.Colowick and N. Kaplan eds., Academic Press, Inc.); HANDBOOK OFEXPERIMENTAL IMMUNOLOGY, Vols. I-IV (D. M. Weir and C. C. BlackwellEds., Blackwell Scientific Publications); and FUNDAMENTAL VIROLOGY, 2ndEdition, Vols. I & II (B. N. Fields and D. M. Knipe, Eds.). Thereference works, patents, patent applications, and scientificliterature, including accession numbers to GenBank database sequences,referred to herein are hereby incorporated by reference in theirentirety. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter.

1. Definitions

Various definitions are made throughout this document. Unless otherwiseindicated, the words have the meaning that would be attributed to thosewords by one skilled in the art. Words specifically defined either belowor elsewhere in this document have the meaning provided in the contextof the present invention as a whole and as are typically understood bythose skilled in the art. Any conflict between an art-understooddefinition of a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter. Headings, however, are provided herein for convenience, andare not to be construed as limiting in any way.

As used herein, the terms “a,” “an,” and “the” refer to the singular andthe plural.

As used herein “GDNF precursor” refers to a glial cell line derivedneurotrophic factor propeptide that contains the N-terminal prosequenceof GDNF joined to the N-terminal sequence of the mature GDNF. The GDNFprecursor may also include the signal sequence.

As used herein, “mature GDNF” refers to glial cell line-derivedneurotrophic factor of vertebrates that has been cleaved from theN-terminal prosequence of the GDNF precursor. The vertebrates may bemammals or non-mammalian vertebrates. In preferred embodiments, themature GDNF is derived from mammals, particularly rodents, non-humanprimates and humans.

As used herein “GDNF prosequence” refers to the N-terminal portion ofthe GDNF precursor that is cleaved from the GDNF precursor uponformation of mature GDNF.

As used herein “proteolytically processed fragment of a GDNF precursor”refers to a fragment of the GDNF precursor formed by proteolyticcleavage of the GDNF precursor.

As used herein, “homologs” refers to analogous sequences in otherorganisms that have substantially the same amino acid sequence of thereference sequence and perform at least one analogous function as thereference sequence. For example, a homolog of GDNF precursor is apolypeptide derived from any animal that has substantial identity at theamino acid level as human, mouse, rat, or chicken peptides.

As used herein, the term “complementary” refers to a nucleic acidsequence, which is able to associate with another nucleic acid accordingto the rules of Watson-Crick base-pairing. For example, an mRNA iscomplementary to a DNA strand of the gene, which encodes it; a DNAsequence and its complementary DNA sequences can form a hydrogen-bondedduplex.

As used herein, the term “polynucleotide” refers to a chain ofnucleotides linked by phosphodiester bonds, including modificationsthereof for improving stability, and includes RNA and DNA.

The term “nucleic acids” as used herein includes, for example, genomicDNA, mRNA, and cDNA.

As used herein, the term “polypeptide” refers to a chain of amino acidresidues linked by peptide bonds.

Any claims to sequences herein encompass those insubstantial alterationsthat can be made to a sequence without effecting function, i.e.,substantially the same sequences. For example, a change in a nucleotidewithin a codon that results in the same amino acid as originally encodedby the codon is “substantially the same sequence.” Also, a conservativeamino acid substitution within the sequence that does not affectfunction is also “substantially the same sequence.”

The term “bind” as used herein refers to the interaction between GDNF orGDNF precursor-derived peptides and their receptors or binding proteins,the binding being of a sufficient strength and for a sufficient time toallow the detection of said binding under the conditions of the assaysdisclosed herein.

The term “about” in reference to a numerical value means 10% of thenumerical value, more preferably 5%, most preferably 2%.

The term “probe” in relation to a polynucleotide or polypeptide includesa probe of sufficient length to signify specific binding. Thepolynucleotides can also be utilized for gene therapy, using both invivo and ex vivo techniques. The polypeptides can also be usedtherapeutically, and to prepare monoclonal and polyclonal antibodiesusing known techniques.

The term “effect” means an alteration or change. An effect can bepositive, such as causing an increase in some material, or negative,e.g., antagonistic or inhibiting.

When referring to a polypeptide, examples of isolated polypeptidesinclude, but are not limited to precursor polypeptides, partially orsubstantially purified polypeptides, peptides produced in a heterologouscell, and synthetic peptides. Preferably the isolated polypeptides ofthe invention are free of additional amino acids in the naturallyoccurring protein and may be substantially purified from culture mediumor chemical precursors when chemically synthesized.

As used herein, “synthesized” refers to polynucleotides and polypeptidesproduced by purely chemical, as opposed to enzymatic, methods. Thesynthesis may be complete or partial synthesis.

As used herein, “activity” refers to any measurable indicia suggestingor revealing binding, either direct or indirect; affecting a response,i.e. having a measurable affect in response to some exposure orstimulus, including, for example, the affinity of a compound fordirectly binding a polypeptide or polynucleotide of the invention, or,for example, measurement of amounts of upstream or downstream proteinsor other similar functions after some stimulus or event.

As used herein, a “neuropeptide” is a polypeptide possessing at leastone neuropeptide activity, and may also have a function or functionsoutside of the nervous system.

As used herein “neuropeptide activity” refers to an activity linked toboth neural cells and non-neural cells and includes, but is not limitedto such activities as (a) receptor binding (e.g., to the cRet orNPY—Y_(i) receptors), (b) stimulation of neural cells to produce aneffect; (c) stimulation of tyrosine kinase activity (d) the ability tomodulate the excitatory synaptic input, for example, into primary cellsin the CA1 area of hippocampal brain slices; (e) immunogenicity thatelicits a neuropeptide-specific immune response in an animal; (f)stimulation of renal tubule regeneration, cessation of hair loss orstimulation of hair growth (g) stimulation or inhibition ofspermatogenesis (h) stimulation of a GDNF-related function in ovariantissue, lung tissue, gastrointestinal tissue, skeletal muscle, cardiactissue and/or adrenal tissue, and/or (h) immunogenicity that elicits animmune response in an animal that cross-reacts with GDNF precursor or ahomolog thereof. Polypeptides that exhibit neuropeptide activityinclude, for example, the neuropeptides of SEQ ID NOs: 9-17, and 5-8.Thus, the term “neuropeptide activity” encompasses functions within thenervous system as well as functions outside of the nervous system.

As used herein “motor disorders” refers to muscle dysfunction,preferably neural-mediated muscle dysfunction. Non-limiting examples ofmotor disorders include such neuropathies as amyotrophic lateralsclerosis (ALS), X-linked spinal and bulbar muscular atrophy (SBMA),chronic inflammatory demyelinating polyneuropathy, and demyelinatingGuillain-Barre syndrome.

As used herein “neurodegenerative disorders” refers to disorderscharacterized by progressive loss of neural function. A non-limitingexample includes Parkinson's disease.

As used herein “neuropathic pain” refers to chronic pain that persistsafter the initial insult to the body has healed. Neuropathic pain may bemarked by a variety of symptoms, including, but not limited tohyperalgesia, and hyperaesthesia.

As used herein “epileptic syndromes” refers to diseases characterized byelectrophysiological disorders of the brain. Non-limiting examplesinclude epilepsy and epileptic seizures.

As used herein “renal disorders” refers to chronic disorders of thekidney and ureters marked by decline in kidney function over time aswell as to acute damage to the kidneys resulting in loss of renalfunction.

As used herein “testicular disorders” refers to disorders of the testisresulting in reduced spermatogenesis.

As used herein “skin disorder” refers to a disorder of the dermal layersthat results in hair loss.

As used herein “cardiac disorders” refers to a disorder of the cardiactissue resulting in abnormal heart rate or rhythm.

As used herein a “pharmaceutically acceptable carrier” refers to acompound or mixture of compounds that are suitable excipients for thedelivery of drugs, antibodies, polynucleotides or polypeptides.

Unless indicated otherwise, as used herein, the abbreviations in lowercase (gdnf) refer to a gene, cDNA, RNA or nucleic acid sequence, whilethe upper case versions (GDNF) refers to a protein, polypeptide,peptide, oligopeptide, or amino acid sequence. Full-length genomic DNAfor human gdnf and cDNA (with excised introns) as well as other nucleicacid molecules encoding a GDNF without the neuropeptide region areencompassed by “gdnf.”

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)₂, and other fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies,anti-anti-idiotypic antibodies, and humanized antibodies.

2. Polypeptides

The neuropeptides of the invention may be derived from human GDNF andGDNF homologs, including, but not limited to mouse, rat and chickenGDNF. The neuropeptides of the invention may be GDNF precursors havingat least one neuropeptide activity. In addition, the polypeptides mayalso have activity outside of the nervous system. The neuropeptides ofthe invention may also be proteolytically processed fragments of GDNFprecursors and homologs that possess at least one neuropeptide activity.As used herein, “proteolytically processed” refers to naturallyoccurring fragments from endogenous enzymatic digestion of the GDNFprotein or homolog, or artificially induced digestion of the GDNFprecursor protein or homolog in vitro.

The proteolytic fragments of the invention comprise amino acid sequencesof GDNF precursor and homologs and homologs that are the result ofenzymatic digestion, containing a protease cleavage site and/or whichhave at least one neuropeptide activity. In preferred embodiments, theproteolytic fragments of the invention comprise the neuropeptides of SEQID NOs: 9-17 and 5-8 (derived from GDNF).

The polypeptides of the invention may be naturally processedneuropeptides that are isolated from cells. Alternatively, the GDNFprecursor protein may be isolated from cells and processed in vitro toobtain the neuropeptides. Alternatively, the neuropeptides of theinvention may be produced using recombinant molecular technology, bydirectly producing the peptides, or by producing larger precursors(including the entire GDNF precursor) and subsequently processing theprotein to produce the neuropeptides. Another alternative is to producethe neuropeptides of the invention chemically.

The polypeptides of the invention include polypeptide sequences thathave at least about 99%, at least about 95%, at least about 90%, atleast about 85%, at least about 80%, at least about 75%, at least about70%, at least about 65%, at least about 60%, at least about 55%, atleast about 50%, or at least about 45% identity and/or homology to thepreferred polypeptides of the invention, the GDNF precursor-derivedneuropeptides or homologs thereof. The preferred polypeptides of theinvention contain the amino acid sequences of SEQ ID NOs: 9-17 and 5-8.In addition, the polypeptides of the invention include polypeptides thatare modified. The neuropeptides of the invention may be glycosylated,lipidated, and/or phosphorylated. Furthermore, chemical modification mayinclude any chemical modification or functional group substitution thatallows the polypeptide to retain at least one neuropeptide activity(such as, but not limited to the ability to elicit an immune responseagainst unmodified neuropeptide), or at least one activity outside ofthe nervous system. In a preferred embodiment, the chemical modificationis α-amidation. Preferred examples of the α-amidated neuropeptides ofthe invention include α-amidated peptides having the amino acid sequenceof SEQ ID NOs: 9, 10, 11, 15, 16, and 17.

The neuropeptides of the invention may also include additions,substitutions or deletions as necessary or helpful in using theneuropeptides of the invention as described herein. For example,neuropeptides may be engineered with an additional N-terminal methionineresidue to aid in recombinant expression of the neuropeptide.Conservative substitutions may also be made in the amino acid sequenceof the neuropeptide provided at least one neuropeptide function isretained by the modified peptide (such as but not limited to the abilityto elicit an immune response against native neuropeptide), or at leastone activity outside of the nervous system is retained by the modifiedpeptide. Conservative substitutions are well known in the art.References for determining conservative substitutions include from WO97/09433 published Mar. 13, 1997, and Lehninger, BIOCHEMISTRY, SecondEdition, Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77, thedisclosures of which are incorporated herein by reference.

Amino acid insertions, deletions and/or substitutions may be introducedinto the neuropeptides of the invention by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Alternatively,the neuropeptides may be chemically synthesized as desired. Preferably,conservative amino acid substitutions are made at one or more amino acidpositions, which are determined to be non-essential, i.e., amino acidresidues which may be substituted without substantial effect onbiological activity.

3. Polynucleotides

The neuropeptides of the invention may be encoded by any polynucleotidesequence that results in the amino acid sequences of SEQ ID NOs: 9-17,and 5-8 or homologs thereof which contain a protease cleavage siteand/or at least one neuropeptide activity. The polynucleotides of theinvention may encode vertebrate GDNF precursor (e.g., human GDNFprecursor (SEQ ID NO:1), mouse GDNF precursor (SEQ ID NO:2), rat GDNFprecursor (SEQ ID NO:3), and chicken GDNF precursor (SEQ ID NO:4) thatis subsequently proteolytically processed into the neuropeptides of theinvention. Due to the degeneracy of the genetic code, a multitude ofnucleic acid sequences may encode the neuropeptides of the invention.The polynucleotides of the invention include all polynucleotides thatencode neuropeptides having a polypeptide sequence derived from a GDNFprecursor or homolog thereof.

The polynucleotides encoding the neuropeptides of the invention may beisolated polynucleotide sequences or fragments thereof, sequences havingcomplementarity to the isolated polynucleotide sequences or fragmentsthereof, or may be part of larger molecules, such as plasmids, forexample. However, the polynucleotides of the invention exclude intactpolynucleotide sequences in their native state as in the chromosome orthe genome of the organism from which it is derived.

One aspect of the present invention is directed to vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules encoding the neuropeptides of the invention. Vectors are usedherein either to amplify DNA or RNA encoding the neuropeptides and/or toexpress DNA which encodes the neuropeptides. As used herein, the terms“vector” and “plasmid” are used synonymously. These refer to nucleicacid molecules capable of transporting another nucleic acid to which ithas been linked. Generally, vectors are capable of autonomousreplication in a host cell into which they are introduced. Other vectorssuch as non-episomal mammalian vectors integrate into the genome of ahost cell upon introduction. These replicate as part of the host genome.Expression vectors contain nucleic acid sequences that direct theexpression of polynucleotide sequences that are operatively linked tothe functional sequences of the expression vector. The invention alsoembraces polynucleotides cloned in viral vectors, including, but notlimited to replication defective retroviruses, adenoviruses andadeno-associated viruses. The expression vectors may also includepolynucleotide sequences that encode a selectable marker (such as a genethat confers drug-resistance), and other sequences to allow formation offusion proteins. Typical drug resistance genes allow resistance totetracyline, kanamycin, and ampicillin. In some embodiments, the fusionprotein aids in protein purification, solubility, or increasedproduction. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) thatfuse glutathione-S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Non-limitingexamples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 1 d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

Expression of proteins may be performed in prokaryotic and eukaryoticcells using appropriate vectors that contain functional sequences thatare functional in the particular cell type. “Functional sequences”refers to polynucleotide sequences that direct a function of geneexpression. Non-limiting examples of functional sequences includepromoters, enhancers, termination sequences, and polyadenylationsequences.

In another embodiment, the neuropeptides are cloned into eukaryoticexpression vectors for expression in suitable eukaryotic cells. Examplesof vectors for expression in yeast S. cerivisae include pYepSec1(Baldari, et al., (1987) EMBO J6:229-234), pMFa (Kurjan and Herskowitz,(1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.). Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195).

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the neuropeptides in a particularcell type. Non-limiting examples of suitable tissue-specific promotersinclude the albumin promoter (liver-specific; Pinkert et al. (1987)Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton(1988) Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banedi et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss (1990)Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman(1989) Genes Dev. 3:537-546).

4. Suitable Host Cells

Suitable host cells for expression of the neuropeptides of the inventioninclude, but are not limited to, prokaryotes, and eukaryotes.Prokaryotic expression vectors are used in conjunction with suitableprokaryotic host cells. The type of prokaryotic cell used is notparticularly limited, but preferred examples include bacteria of thegenera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, andStaphylococcus.

Eukaryotic expression vectors are used in conjunction with eukaryoticcells. Preferred host cells include, but are not limited to, yeastcells, insect cells, HeLa cells, Chinese hamster ovary cells (CHOcells), African green monkey kidney cells (COS cells), human 293 cells,and murine 3T3 fibroblasts. Preferred eukaryotic host cells include, butare not limited to, the genera Saccharomyces, Pichia, Kluveromyces;Spodoptera frugiperda cells, Drosophila Schneider cells, HeLa cells, CHOcells, COS cells, 293 cells and 3T3 cells.

5. Antibodies

The antibodies of the invention include antibodies that specificallybind the neuropeptides of the invention. As such, the origin and isotypeof the antibodies is not particularly limited. The antibodies may beraised in any antibody-producing animal and may be of any isotype,including, but not limited to IgA, IgD, IgM, IgG, IgE, monoclonalantibodies, chimeric antibodies, grafted antibodies, humanizedantibodies, superhumanized antibodies, provided the antibodiesspecifically bind to at least one neuropeptide of the invention. Theantibodies of the invention also include fragments of antibodies thatspecifically bind at least one neuropeptide of the invention, singlechain antibodies, and anti-idiotypic antibodies generated by immunizingan animal with any of the specific anti-neuropeptide antibodies of theinvention.

The term “monoclonal antibody” as used herein, refers to animmunoglobulin molecule that contains a single species of an antigenbinding that has affinity for a particular epitope (of a givenneuropeptide of the invention).

The anti-idiotypic antibodies include, but are not limited to internalimage anti-idiotypic antibodies. The antibodies and anti-idiotypicantibodies of the invention may be generated by methods which are wellknown in the art, and which may be found in a variety of references,such as the HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., Blackwell Scientific Publications).

Methods of immunizing animals and generating antibodies, monoclonalantibodies and anti-idiotypic antibodies are well established and knownto those of ordinary skill in the art. U.S. Pat. No. 4,946,778 describesa method for the production of single chain antibodies, and isincorporated herein by reference. Non-human antibodies can be“humanized” by techniques well known in the art (e.g., U.S. Pat. No.5,225,539). Grafted antibodies are those that are humanized by graftingnon-human CDRs onto a human antibody constant region, or grafting thenon-human CDRs onto a consensus antibody framework sequence. Furtherchanges can then be introduced into the antibody framework (such asthose derived from the same organism as the origin of the CDR) tomodulate affinity or immunogenicity (“superhumanized” antibodies).

Internal image anti-idiotypic antibodies are raised against antibodiesagainst the neuropeptide that mimic the neuropeptide. These antibodiesand methods of preparing them are described in Bona and Kohler (1984)ANTI-IDIOTYPIC ANTIBODIES AND INTERNAL IMAGE, IN MONOCLONAL ANDANTI-IDIOTYPIC ANTIBODIES: PROBES FOR RECEPTOR STRUCTURE AND FUNCTION,Venter J. C., Frasser, C. M., Lindstrom, J. (Eds.) NY, Alan R. Liss, pp141-149, 1984, which is incorporated herein by reference.

The antibodies of the invention are useful for detecting theneuropeptides of the invention in situ, in assays such asradioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),Western blots, and other well known assays utilizing specificantibodies. The antibodies of the invention are also useful astherapeutics. Such antibodies may be administered to an animal tomodulate the effects of a given neuropeptide. Modulating the effects ofneuropeptides may include positively or negatively influencing theeffect of the neuropeptide. For example, an internal imageanti-idiotypic antibody may be administered to increase the effect ofthe neuropeptide by mimicking the neuropeptide. Antibodies directedagainst the neuropeptide may act to inhibit the effect of theneuropeptide.

Other antibodies of the invention include portions of antibodies thatspecifically bind the neuropeptides of the invention and which may befused to other proteins, or portions thereof.

6. Methods of Identifying Receptors

The neuropeptides of the invention may be used as probes to identify thereceptors of the neuropeptides. To identify the neuropeptide receptors,a polypeptide comprising at least a portion of the neuropeptides of theinvention (e.g., SEQ ID NOs: 9-17, 5-8) can be used as a probe inscreening neuronal and non-neuronal cell lines using techniques wellknown in the art. As a non-limiting example, binding assays can beperformed as follows. Cells can be incubated with a labeledneuropeptide, such as an iodinated neuropeptide, in Dulbecco's phosphatebuffered saline and 2 mg/ml bovine serum albumin (BSA) on MilliporeHydrophilic Durapore 96-well filtration plates. Following two hours ofvigorous shaking at 4° C., the cells are washed twice with ice-coldbinding buffer under a vacuum. Dried filters are liberated and bound¹²⁵I-neuropeptide is quantified in a gamma counter. Non-specific bindingis determined by addition of 500-fold excess of cold ligand to thebinding mixtures.

For affinity labelling, labeled neuropeptides (e.g., iodinatedneuropeptides) are bound to monolayer cultures of primary neurons orcell lines. Prior to binding, cells are cultured for 48 hours in thepresence of NGF on polyornithine/laminin coated dishes. Plated cells areincubated with 10 ng/ml ¹²⁵I-neuropeptide at 4° C. in binding buffer asdescribed above. Ligand/receptor complexes are chemically cross-linkedfor thirty minutes at room temperature using either disuccinimidylsuberate (DSS) or 1-Ethyl-3(−3-dimethylaminopropyl)-carbodiimidehydrochloride (EDAC) (Pierce Chemical, Rockland, Ill.). Followingquenching of the cross-linking reactions, cells are washed twice with 10mM Tris/HCI buffered saline, 2 mM EDTA, 10% glycerol, 1% NP-40, 1%Triton X-100, 10 μg/ml leupeptin, 10 μg/ml antipain, 50 μg/ml aprotinin,100 μg/ml benzamidine hydrochloride, 10 μg/ml pepstatin and 1 mM PMSF(proteinase inhibitors from Sigma). Cleared lysates are boiled for 5 minin SDS/β-mercaptoethanol buffer, fractionated by SDS/PAGE on 4-20%gradient electrophoresis gels, and visualized by autoradiography.Molecular weights for the receptors can be obtained by subtracting theweight of the neuropeptide from the estimated molecular weights ofcross-linked complexes visualized by SDS/PAGE.

For affinity measurements of cross-linked complexes, cells are incubatedon plates as above in the presence of increasing amounts of unlabelledneuropeptide. The samples are fractionated by gradient SDS/PAGE, gelsare then dried and specific bands excised according to molecular weightsdetermined from autoradiograms, and counted in a gamma counter.

Other methods of identifying receptors include affinity chromatographyusing immobilized neuropeptides. Once a receptor candidate isidentified, the protein can be subjected to amino acid sequencing orpartial amino acid sequencing using standard procedures such as Edmunddegradation, and degenerate oligonucleotide probes may be synthesizedthat correspond to the possible nucleic acid sequence encoding thisregion of the receptor candidate, and the oligonucleotides may be usedas probes to screen cDNA libraries, or may be used as primers in apolymerase chain reaction (PCR) to amplify the appropriate portion ofthe genomic sequence of the receptor candidate. DNA sequencing may beemployed to determine the molecular sequence of the receptor candidate.

Other methods of screening for receptors or modifications of the abovemethods, involving routine experimentation, would be recognized by oneof ordinary skill in the art. Modifications of the methods presentedherein may be made without departing from the spirit and scope of theinvention.

Without being bound by any particular theory of operation, it isbelieved that the GDNF precursor derived neuropeptides of the inventioninteract with the receptor for GDNF (cRet and/or GDNF family receptorα1). By binding to cRet, the GDNF precursor-derived neuropeptides of theinvention may stimulate these tyrosine kinase receptors to inducebiological responses similar to that induced by the binding of GDNF or ahomolog of GDNF. The neuropeptides may, therefore, be used assubstitutes for GDNF in therapeutic contexts, as probes, and in assays.Therefore, the neuropeptides of the invention may be used in any utilityknown for GDNF in which GDNF interacts with one of its receptors.Specific examples of such utilities are described in more detail herein,but include use as therapeutics in neuronal disorders and non-neuronaldisorders, including, but not limited to renal disorders, cardiacdisorders, skin disorders, and testicular disorders.

The peptides of the invention may also interact with other knownreceptors. For example, and not by way of limitation, two of theneuropeptides of the invention (SEQ ID NO:9 and SEQ ID NO:11) modulatethe excitatory synaptic input into primary cells in the rat CA1 as shownby acute rat hippocampal brain slice preparation studies (see Example5). Furthermore, the homology between the peptides (shown in Example 2)SEQ ID NO:10, SEQ ID NO:11 and Peptide YY (PYY) suggests that thepeptides of the invention may interact with the same receptors asNeuropeptide Y (NPY) and PYY.

Glutamate is a potent excitatory neurotransmitter. Studies of NPY actionin the hippocampus using brain slices in vitro suggest that NPY has apotent inhibitory action on the release of glutamate in the hippocampusthrough the NPY—Y2 receptors (Greber, S. et al (1994) Br. J. Pharmacol.113(3):737-40). In other studies, it has been shown that NPY actspresynaptically in the hippocampal CA1 region to reduce excitatory inputto the pyramidal neurones (Colmers, W. F. et al. (1987) J. Physiol.383:285-299). It has been suggested that NPY inhibits excitatorysynaptic transmission at the Schaffer collateral-CA1 synapse by actingdirectly at the terminal to reduce a Ca2+ influx (Colmers, W. F. et al.(1988) J. Neurosci. 8:3827-2837).

The neuropeptides of the invention are shown herein to increase ordecrease synaptic transmission and are thus useful for modulatingexcitatory input into pyramidal neurons and lessen or increase glutamaterelease and subsequent excitatory effects. This discovery findsparticular utility in modulating excitatory effects (presumably mediatedthrough NPY and/or PYY receptors). As such, the peptides may beadministered to a subject to modulate immune responses, gastrointestinalmotility, pancreatic and adrenal function, eating activity, circadianrhythm, arousal, blood circulation and pressure, and cell proliferationin normal and malignant tissues.

The data indicate that the neuropeptides of the invention are useful formodulating biological effects involving NPY and PYY receptors. Therapiesdirected to modulation of NPY and PYY receptors are embraced by theinvention. The peptides of the invention may be administered to subjectsfor the treatment of such disorders related to excitatoryneurotransmission as neuropathic pain, epilepsy, stroke and brain damagein premature infants.

In a preferred embodiment, the peptide of SEQ ID NOs: 9 and/or 10 and/or11 is administered to subjects for the treatment of such disordersrelated to excitatory neurotransmission as neuropathic pain, epilepsy,stroke and brain damage in premature infants.

The peptides of the invention may also be used in vitro to modulateexcitatory neurotransmission.

7. Expression of Neuropeptides

As described above, expression of the polynucleotides encoding theneuropeptides of the invention may be effected by inserting thepolynucleotides encoding the neuropeptides into an appropriateexpression vector and introducing the polynucleotides into anappropriate cell type. Introduction of nucleic acid into cells may be byany method known in the art, including, but not limited to transfection(e.g., calcium phosphate precipitation, electroporation, gene gun“biolistic” techniques), transformation, or transduction.

The neuropeptides of the invention may also be expressed in vitro byusing such a system as a rabbit reticulocyte lysate (RRL) system, awheat germ agglutinin system, or any other in vitro expression systemknown in the art. Kits are available for in vitro translation systemsand include RRL and wheat germ agglutinin kits produced by Promega.Methods of performing in vitro translation are provided by themanufacturer.

The expression of neuropeptides in Cos cells is disclosed below. Theneuropeptides of the invention can also be expressed in other systemsknown in the art, such as bacteria, baculovirus or DrosophilaSchneider-2 cells. A method for expressing proteins using a baculovirusis described in PCT/US96/18197 (incorporated herein by reference).Bacterial expression can be performed following techniques well known inthe art, for example, Peränen J. et al. (1996) Anal. Biochem.,236:371-373 (incorporated by reference herein). An easy approach to useof Schneider-2 cells is offered by the Invitrogen Drosophila Expressionsystem.

8. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject manifesting at least one sign of a motordisorder, neurodegenerative disease, epileptic syndrome, renal disorder,hair loss, and/or neuropathic pain. The invention also provides for thetreatment of disorders associated with increased or decreased expressionof neuropeptides derived from GDNF precursor or a homolog thereof. Thecompositions that are useful for treatment of these disorders may betherapeutic, or prophylactic. Diseases and disorders that arecharacterized by increased (relative to a subject not suffering from thedisease or disorder) levels or biological activity may be treated withcompositions that antagonize (i.e., reduce or inhibit) activity.Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with agonists (i.e., compounds thatincrease activity). The compositions that are suitable for use in theinvention include, but are not limited to GDNF precursor-derivedneuropeptides or analogs, derivatives, fragments or homologs thereof;anti-neuropeptide antibodies; nucleic acids encoding a GDNF precursor orhomolog-related neuropeptide; modulators (i.e., inhibitors, agonists andantagonists, including additional peptide mimetic of the invention orantibodies specific to a peptide of the invention) that alter theinteraction between a GDNF precursor or homolog-related neuropeptide.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, aGDNF or homolog-derived neuropeptide, or analogs, derivatives, fragmentsor homologs thereof; an agonist that increases bioavailability; oragents that stimulate GDNF expression and/or proteolytic processing.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of a GDNFor homolog-derived neuropeptide). In general, GDNF precursor-derivedneuropeptides may be quantified in comparison to normal tissue. Inaddition, the amount of GDNF precursor or homologs thereof may beanalyzed in relation to normal cells. RNA encoding GDNF precursor orhomolog may also be used as an index of GDNF precursor expression whichcan be used to extrapolate the amount of GDNF precursor-derivedneuropeptides (or homologs thereof) resulting from the precursors.

Methods that are well-known within the art to analyze nucleic acid andprotein include, but are not limited to, immunoassays (e.g., by Westernblot analysis, immunoprecipitation followed by sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.)and/or hybridization assays to detect expression of mRNAs (e.g.,Northern assays, dot blots, in situ hybridization, reverse transcriptasepolymerase chain reaction (rtPCR), etc.).

In one aspect, the invention provides a method for preventing a diseaseor condition associated with an aberrant GDNF precursor-derivedneuropeptide (or homologs thereof) expression or activity, byadministration of a composition that modulates expression of GDNFprecursor-derived neuropeptides (or homologs thereof) or at least oneneuropeptide activity. Compositions that modulate expression,proteolytic cleavage or activity of the GDNF precursor-derivedneuropeptides of the invention may be used as treatments for conditionsrelated to aberrant GDNF precursor-derived neuropeptide (or homologthereof) expression or activity. Depending on the type of neuropeptideaberrancy, an agonist or antagonist can be used for treatment.

Another aspect of the invention pertains to methods of modulating theexpression or activity of the neuropeptides of the invention bycontacting a cell with a composition that modulates one or more of theactivities of the neuropeptides of the invention. Such a composition mayinclude a neuropeptide of the invention, or a fragment or analogthereof; an anti-neuropeptide antibody of the invention; a fusionprotein containing a neuropeptide of the invention or fragment or analogthereof; at least a portion of an anti-neuropeptide antibody thatspecifically binds to a neuropeptide of the invention (which isoptionally fused to another protein, or portion thereof); and/or apolynucleotide encoding a neuropeptide of the invention, or fragment,fusion protein, or analog thereof. The methods of the invention fortreating a subject may be performed in vivo, ex vivo, or in vitro. Theamounts of the therapeutic will vary according to the method and activeingredient used. Typically, the amount of neuropeptide, antibody ormodulatory agent used will be an amount sufficient to achieve thedesired effect without causing any untoward effects in the subject.

In some embodiments of the invention, peptides are administered tosubjects to modulate the effects associated with PYY and/or NPYreceptors. The neuropeptides of the invention may be used to up-regulateor down-regulate these effects. For example, and not by way oflimitation, the neuropeptides of the invention may be used to competewith PYY and NPY to down-regulate the effects of NPY and/or PYY.Alternatively, the neuropeptides of the invention may be administered tostimulate an effect by interacting with NPY and/or PYY receptors. Inpreferred embodiments, the neuropeptides of the invention areadministered to subjects as a means to cause vasoconstriction, cardiacstimulation, or inhibition of noradrenaline and/or renin release. Inpreferred embodiments of the invention, a peptide of the sequence shownin SEQ ID NO:9 and/or SEQ ID NO:10 and/or SEQ ID NO:11 is administeredto a subject to modulate activity associated with NPY and/or PYYreceptors.

Peptides of the invention may be administered alone, or n combinationwith at least one pharmaceutically acceptable carrier for the treatmentof disorders associated with excitatory neurotransmission. Suchdisorders include, but are not limited to epilepsy, stroke and braindamage.

In preferred embodiments, the neuropeptides of the invention may be usedto treat such diseases and disorders as renal disorder (e.g., thecompositions of the invention are useful in promoting tubularregeneration following renal damage), motor diseases, neurodegenerativediseases, neuropathic pain and other neurological disorders such asepileptic syndromes. The neuropeptides of the invention may also be usedto treat disorders of the enteric nervous system and may be used totreat skin disorders such as hair loss as a result of apoptosis-drivenhair follicle involution.

The compositions of the invention may be formulated for enteric andparenteral use. Such formulations include at least one pharmaceuticallyacceptable carrier for delivering the neuropeptides of the invention orfragments thereof. The formulations may be made for delivery for anyroute of administration, including oral, nasal, rectal, topical,intramuscular, intradermal, interperitoneal, and subcutaneous routes.The compositions may also be formulated as inhalants.

The specific application may determine the optimal route ofadministration. As an example, but not by way of limitation, acomposition of the invention containing at least one neuropeptide of theinvention may be formulated as a topical composition when it is to beused for prevention of hair loss. As another non-limiting example,compositions for treating renal disease may be formulated as an oralcomposition or injectable composition. The person of ordinary skill inthe art is acquainted with formulation strategies for optimal deliveryof pharmaceuticals to particular tissues for optimal efficacy and easeof administration and would know how to modify the compositions for use.Such modifications are within the scope of the invention.

The invention is further described in the Examples as set forth below.Example 4 is a prophetic example. The examples are provided merely asillustrations of the embodiments of the invention and are not to beconstrued as limiting the scope of the invention which is set forth inthe appended claims.

EXAMPLES Example 1 Identification of Vertebrate GDNF-DerivedNeuropeptides

Analysis of the sequence of the human GDNF precursor revealed additionalPC processing sites within the proregion (SEQ ID:s 5-8) and also in theamino-terminal half of the mature GDNF (SEQ ID:s 20-23) (FIG. 1).Processing at the indicated sites would result in formation of short(4-17 amino acids) peptides, and after successive action ofcarboxypeptidase E/H and peptidylglycine α-amidating monooxygenase PAM(Eipper B. et al. (1993) Prot. Sci. 2:489-497) three of them would yieldamidated peptides (Table 1).

The cysteine knot of GDNF implicates strong structure-functionrelationship in GDNF as the cysteine-bonding pattern of the substratesaffects the accessibility of putative processing sites. However, all theimplicated peptide products originate either from the propeptide, whichis separated from the folded GDNF precursor reaching the convertaseresponsible for GDNF maturation at late stage of the secretion pathway,or from the N-terminal part of the mature GDNF preceding the firstcysteine. None of the implicated processing sites would thus be buriedinto the knot, and as the peptides do not contain Cys residues they canbe released from the precursor by endopeptidase cleavage. Furthermore,formation of any of the suggested peptides would not affect the activityof mature GDNF (Baloh et al. (2000) J. Biol. Chem. 275(5), 3412-3420).

Other precursors of the GDNF family of neurotrophic factors (artemin,neurturin, persephin) do not contain similar good candidate sites forpeptide production, and none of the putative weak processing sites wouldyield amidated peptides. Instead, human GDNF precursor contains severalwell-conserved substrate sites for furin/PC convertases and otherenzymes required for release of neuropeptide-like products. SEQ IDfunctional NO: name name sequence species  9 PEP1 Neuron FPLPA(a) human,Related mouse, rat Peptide NRP 10 hPEP2 PPEAPAEDRSL(a) human 11 rPEP2Brain LLEAPAEDHSL(a) mouse, rat Excitatory Peptide BEP 12 hPEP3SPDKQMAVLP human 13 rPEP3 SPDKQAAALP mouse, rat 14 SPDKQTPIFS chicken 15hPEP4 ERNRQAAAANPENS human RGK(a) 16 rPEP4 ERNRQAAAASPENS mouse, ratRGK(a) 17 ERNRQSAATNVENS chicken SKK(a)

Table 1 shows the sequences of the GDNF precursor-derived neuropeptidesof human, mouse, rat and chicken. “a” indicates alpha amidation. Thepeptides shown are from human, mouse and rat (SEQ ID NO:9); human (SEQID NO:10); mouse and rat (SEQ ID NO:11); (SEQ ID NO:12); mouse and rat(SEQ ID NO:13); chicken (SEQ ID NO:14); human (SEQ ID NO:15); mouse andrat (SEQ ID NO:16); and chicken (SEQ ID NO:17). In the PEP-nomenclatureof the peptides, rodent or human origin is indicated by the preceedingr- or h-, respectively, and the numbers refer to the order of thepredicted peptides along the respective GDNF precursor from the N- toC-terminus.

Example 2 Sequence Analysis of the Human GDNF-Derived Neuropeptides

GDNF-derived neuropeptides were analyzed for similarity to other, knownneuropeptides based on homology at the amino acid level.

Neuropeptides derived from the proregion of GDNF (SEQ ID NO:10 and SEQID NO:11) are highly homologous to a recently identified member of theneuropeptide Y (NPY) family. This new neuropeptide, the human peptideYY2 (hPYY2) (SEQ ID NO:18) (Couzens et al. (2000) Genomics 64 (3),318-323), is the closest human equivalent of the long known bovinePYY2/seminaplasmin. An alignment of SEQ ID NO:10 and SEQ ID NO:11 withhPYY2 (SEQ ID NO:18) yields: SEQ ID NO:10  P--PEAPAEDRSL (a) *  **** **  * SEQ ID NO:18 hPYY2 YPIKPEAPGEDAFL (a)      *** **  * SEQID NO:11    LLEAPAEDHSL (a)

The amidation (a) of the hPYY2 is not certain, but identity betweenhPYY2 and SEQ ID NO:10 is 57-63%, depending on the region compared. Thelower percentage relates to identity between SEQ ID NO:10 and the wholehPYY2. The higher percentage refers to identity between SEQ ID NO:10 andthe eleven C-terminal amino acids of hPYY2.

The homologous region can also be found in other PYY and NPY-familypeptides, but hPYY2 is the shortest member of this family and besthomolog of SEQ ID NO:10 and SEQ ID NO:11.

NPY and its receptors (Y_(i)) are both expressed in hippocampalinterneurones. In the rat hippocampus, application of NPY inhibitsexcitatory transmission in the CA1 area (Colmers et al. (1987) J.Physiol. 383:285-299; Colmers et al. (1988) J. Neurosci. 8:3827-3837.Activation of Y_(i) by NPY inhibits presynaptic voltage-operated calciumchannels, resulting in a decrease in presynaptic calcium levels andconcomitantly reduced transmitter release (Qian et al. (1997) J.Neurosci. 17:8169-8177). The homology between peptide YY (PYY) and theGDNF-derived peptides SEQ ID NO:10 and SEQ ID NO:11 suggests that theseGDNF-derived peptides (SEQ ID NO:s 10 and 11) could act on the samereceptors (Y_(i)) as NPY and PYY. This hypothesis gains strength fromthe experiments shown in Example 3 (below).

Example 3 Biological Effects of GDNF Precursor-Derived Neuropeptide

The human GDNF-derived neuropeptide that is closest to the N-terminus ofthe GDNF precursor is shown in SEQ ID NO:9. The biological effects ofthe SEQ ID NO:9 and SEQ ID NO:11 neuropeptides were studied using anacute rat hippocampal brain slice preparation. Wistar rats (P28-P44)were anesthetized with ketamine and medetomidine and transcardiallyperfused with ice-cold artificial cerebrospinal fluid (ACSF) containing(in mM) 124 NaCl, 5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 MgSO₄, 2 CaCl₂, and10 glucose, continuously gassed with 5% CO₂-95% O₂. After decapitationthe brains were exposed and 400-μm-thick transverse hippocampal slicescut with a vibratome while in high-sucrose solution containing (in mM)230 sucrose, 3 KCl, 8 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, and 25glucose. The slices were allowed to recover in continuously gassed ACSFat 32° C. for 30 min and at room temperature for at least 30 min beforeuse. While recorded, the slices were held at 32° C., continuously gassedwith 5% CO₂-95% O₂ and perfused with ACSF at 3 ml/min. A bipolarstimulation electrode was placed in the stratum radiatum in the CA1area. Extracellular responses were evoked by single stimuli (submaximalamplitude, duration 100 μs, frequency 0.05 Hz) and recorded from thestratum pyramidale using glass microelectrodes with a tip resistance of2-6 MΩ when filled with extracellular solution containing (in mM) 150NaCl, 5.4 KCl, 1 MgCl₂, 1.8 CaCl₂, and 5 Hepes, pH adjusted to 7.4 withNaOH. Results were analyzed using the WCP program (kindly provided byDr. J. Dempster, University of Strathclyde, UK). Peptides (synthetic,unlabeled, with no additional residues but with C-terminal amidation)and N-ethylmaleimide (NEM; Sigma, St. Louis, Mo.), which selectivelyblocks signaling via pertussis-toxin sensitive G_(i/o)-proteins in theCNS (Morishita et al (1997) J. Neurosci. 17(3): 941-950) were deliveredvia bath perfusion at 3 ml/min. NEM has complex effects on baselinesynaptic transmission and has been reported to cause either first anincrease and then a decrease on pEPSPs (Morishita et al (1997) J.Neurosci. 17(3): 941-950) or only an increase (Tang and Lovinger (2000),J. Neurophysiol. 83(1): 60-69). In our experiments, NEM increased thestimulus-evoked responses in every slice recorded.

Extracellular population excitatory postsynaptic potentials (pEPSPs) andpopulation spikes (pSPs) evoked by 0.05 Hz electrical stimulation of theSchaffer collateral fibres were recorded in hippocampus area CA1 ofslices taken from P28-P35 rats. The pEPSPs and pSPs were significantlyincreased by 30-40% upon superfusion with 10-50 nM the SEQ ID NO:11neuropeptide, suggesting that SEQ ID NO:11 neuropeptide enhances theexcitatory synaptic input into primary cells in the CA1 area. SEQ IDNO:11 also increased presynaptic excitability, seen as an averageincrease of 34% in the presynaptic fiber volley. SEQ ID NO:9 attenuatedthe synaptic transmission, as demonstrated by the significant decreaseof presynaptic fiber volley and pSPs (−24%). These effects were fully oralmost fully reversed upon removal of the SEQ ID NO:9 or SEQ ID NO:11neuropeptides from the superfusate.

The observation that the effects of SEQ ID NO:s:9 and 11 on thehippocampal CA1 synapses resembles or opposes those observed for NPY andPYY suggests that the neuropeptides of the invention may act on the samereceptors as NPY and/or PYY. The NPY receptors are coupled toG-proteins, preferentially to G_(i/o) (Wess, 1998). Application of SEQID NO: 11 in the presence of a G_(i/o)-blocking agent had no effect onexcitability (FIG. 4 B, C, D), strongly suggesting that the excitatoryaction of SEQ ID NO:11 is also mediated via a presynapticG_(i/o)-coupled receptor. The activity of SEQ ID NO:11 or SEQ ID NO:10in human brain could not be tested. However, gdnf expression persists inthe hippocampal formation until adulthood also in man (Serra et al.(2002), Brain Res. 928(1-2): 160-164).

Example 4 Detection of Secreted GDNF Precursor-Derived Peptides fromConditioned Media

Secreted GDNF precursor-derived peptides can be detected fromconditioned media by transfecting cells (such as PC12, Neuro2A) withhuman pro-GDNF-encoding polynucleotides (e.g., mRNA transcribed in vitrofrom pSFV1-hgdnf construct (pSFV1: Gibco BRL)) and metabolicallylabelling the proteins of the transfected cells with a ¹⁴C-amino acidmixture. The medium from the cells can be collected and analyzed bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) inTris-Tricine buffers and visualized by standard procedures.Alternatively, secreted GDNF precursor-derived peptides can be detectedfrom the growth media of cells, which are endogenously producing GDNF,such as the rat glial cell line C6 (ATCC CCL-107) (Suter-Crazzolara andUnsicker, (1996) Brain Res. Mol. Brain Res. 41(1-2): 175-182). Cells aregrown to confluency in DMEM, 10% fetal calf serum before the media ischanged to serum-free OPTIMEM I (Invitrogen, Carlsbad, Calif.)supplemented with 0.1% BSA. After two days the serum-free media iscollected and Complete-Mini protease inhibitor cocktail tablets (Roche,Basel, Switzerland) added. Collected media can directly be subjected tomass spectrometric analysis with MALDI-TOF and ion-trap ESQUIRE, or befurther purified with centrifugation through YM-10 and/or YM-3 columns(Millipore, Bedford, Mass.) and subsequent dialysis against ultra-purewater with MWCO 100 Da membrane (Spectrum Laboratories, RanchoDomingues, Calif.). If required, samples can be lyophilized andresuspended to 50× concentration in water.

Example 5 Binding of Iodinated Peptides to Neuronal Tissues

The binding of I¹²⁵-labeled peptides to embryonic and adult rodenttissues was studied in vitro. Chemically synthesized peptides containingan extra N-terminal tyrosine (Sigma-Genosys, Cambridge, UK) were labeledwith lactoperoxidase (Trupp et al. (1995) J. Cell Biol. 130(1):137-148). Specific activities obtained for each were 0.5-2.5×10¹⁴cpm/mmol, except for SEQ ID NO:9 10¹³ cpm/mmol. From both E17 Wistar ratand E15 mouse embryos, kidneys, gut, pancreas and spinal cord weredissected. In addition, testes, ovaries, brain and adrenal glands of theembryonic mice were taken. The binding was performed and paraffinsections of embryonic tissues were processed as described (Partanen etal. (1987) Dev Biol 120(1): 186-197). Freshly cut adult rat brain slicesof 1 mm were cut and incubated in 0.5-1 nmol/ml of ¹²⁵I-peptides, washedand processed for paraffin sections. Kodak (Rochester, N.Y.) NTB-2emulsion was used for autoradiography and the samples were exposed from3 to 12 weeks at +4° C. The sections were developed with Kodak D19 andstained with hematoxylin.

For immunohistochemical staining of tissues, paraffin sections weremounted with Immumount (Shandon, Pittsburgh, Pa.). Sections were stainedwith an anti-class III β-tubulin antibody (Tuj1; Covance Inc.,Princeton, N.J.), which recognizes selectively neuronal cells. Stainingwas performed with Vectastain kit (Vector Laboratories, Burlingame,Calif.) according to manufacturer's instructions, except that theprimary antibody (1:300) was incubated in +4° C. o/n.

Only SEQ ID NO:s 9 and 11 resulted in specific binding. SEQ ID NO:11(FIG. 3 A-B), but not SEQ ID NO:10 (FIG. 3 C-D), displayed extensivebinding exclusively in adult rat brain (FIG. 3 A-D). Binding of SEQ IDNO:9 was restricted to embryonic mouse tissues outside the CNS.Distribution of the I¹²⁵-labelled SEQ ID NO:9 binding (FIG. 3 E, G)closely overlapped the neuronal β-tubulin staining (FIG. 3 F, H) both inthe gut (FIG. 3 E, F) and the kidney capsule (FIG. 3 G, H) (FIG. 3 E-H).Although the autoradiographic detection of peptide binding in tissuedoes not allow localization of signal at the cellular level, itindicates that SEQ ID NO:9 was bound into neuronal or adjacent cells.The peptide-specificity of the binding patterns was demonstrated by thecontrol peptide I¹²⁵-labelled SEQ ID NO:15 in embryonic mouse testis(FIG. 5A) and gut (FIG. 5B).

In testis, I¹²⁵-labelled SEQ ID NO:9 binding to the interstitium (I) wascomplementary both to the expression of GDNF and to the localization ofneurons, detected by Tuj1 staining, within the seminiferous tubules (J).

Effective Concentrations of Peptides are Physiologically Relevant

The nanomolar effective concentrations of SEQ ID NO:11 (FIG. 6) areremarkably lower than the 1 μM NPY and PYY₃₋₃₆ levels necessary forinhibition of synaptic transmission (Qian et al (1997), J. Neurosci.17(21), 8169-8177). In fact, they are within the same range as thenanomolar GDNF concentrations required for promotion of uretericbranching (Sainio et al. (1997), Development 124(20), 4077-4087) ormaintenance of primary neurons (U. Arumäe, personal communication). AsSEQ ID NO:11 is synthesized in equimolar amounts to GDNF in vivo, thephysiological concentration of SEQ ID NO:11 in vivo can reach the levelrequired for synaptic excitation. This also suggests that the peptidesof the invention may be more potent than NPY or any other knownneuropeptide in modulating the excitatory response.

Various modification of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

1. An isolated polypeptide comprising a GDNF precursor or homologthereof wherein said polypeptide comprises at least one neuropeptideactivity.
 2. The isolated polypeptide of claim 1 wherein saidpolypeptide is a native GDNF precursor isolated from cells.
 3. Theisolated polypeptide of claim 1 wherein said polypeptide isrecombinantly produced.
 4. The isolated polypeptide of claim 1 whereinsaid polypeptide is chemically synthesized.
 5. The isolated polypeptideof claim 1 wherein said GDNF precursor comprises a vertebrate GDNFprecursor fragment or homolog thereof.
 6. The isolated polypeptide ofclaim 5 wherein said vertebrate is selected from the group consisting ofbird, rodent, non-human primate and human.
 7. The isolated polypeptideof claim 1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. 8.An isolated polypeptide comprising a fragment of a GDNF precursor orhomolog thereof wherein said polypeptide comprises at least oneneuropeptide activity.
 9. The isolated polypeptide of claim 8 whereinsaid polypeptide is a proteolytically processed fragment.
 10. Theisolated polypeptide of claim 9 wherein said polypeptide is a nativeprocessed fragment of a GDNF precursor isolated from cells.
 11. Theisolated polypeptide of claim 8 wherein said polypeptide isrecombinantly produced.
 12. The isolated polypeptide of claim 8 whereinsaid polypeptide is chemically synthesized.
 13. The isolated polypeptideof claim 8 wherein said GDNF precursor comprises a vertebrate GDNFprecursor or homolog thereof.
 14. The isolated polypeptide of claim 13wherein said vertebrate is selected from the group consisting of bird,rodent, non-human primate and human.
 15. The isolated polypeptide ofclaim 8 wherein said neuropeptide is α-amidated.
 16. The isolatedpolypeptide of claim 8 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQID NO:17.
 17. The isolated polypeptide of claim 15 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 18. An isolated nucleic acidcomprising a polynucleotide sequence encoding a GDNF precursor orhomolog thereof, wherein said polynucleotide sequence encodes apolypeptide comprising at least one neuropeptide activity.
 19. Theisolated nucleic acid molecule of claim 18 wherein said GDNF precursoror homolog thereof comprises a vertebrate GDNF precursor.
 20. Theisolated nucleic acid of claim 18 wherein said GDNF precursor comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
 21. An isolated nucleicacid comprising a polynucleotide sequence encoding fragment of a GDNFprecursor or homolog thereof, wherein said polynucleotide sequenceencodes a polypeptide comprising at least one neuropeptide activity. 22.The isolated nucleic acid of claim 21 wherein said GDNF precursor orhomolog thereof comprises a vertebrate GDNF precursor.
 23. The isolatednucleic acid of claim 21 wherein said GDNF precursor comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 24. An antibody comprising atleast a portion of an immunoglobulin that specifically binds to apolypeptide comprising a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 25. Theantibody of claim 24 wherein said GDNF precursor comprises a vertebrateGDNF precursor.
 26. The anti-neuropeptide antibody of claim 24 whereinsaid antibody is a monoclonal antibody.
 27. The antibody of claim 24wherein said antibody specifically binds to a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
 28. An antibody comprising atleast a portion of an immunglobulin that specifically binds to afragment of a GDNF precursor or homolog thereof, wherein said fragmentcomprises at least one neuropeptide activity.
 29. The antibody of claim28 wherein said GDNF precursor comprises a vertebrate GDNF precursor orhomolog thereof.
 30. The antibody of claim 28 wherein said antibody is amonoclonal antibody.
 31. The antibody of claim 28 wherein said antibodyspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, and SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, and SEQ ID NO:17.
 32. A vector comprising a polynucleotidesequence encoding a polypeptide wherein said polypeptide comprises aGDNF precursor or homolog thereof and comprises at least oneneuropeptide activity.
 33. The vector of claim 32 wherein said vector isan expression vector that directs expression of said polynucleotidesequence encoding said polypeptide.
 34. The vector of claim 32 whereinsaid polynucleotide sequence encodes a polypeptide comprising apolypeptide sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
 35. A vector comprising apolynucleotide sequence encoding a polypeptide wherein said polypeptidecomprises a fragment of a GDNF precursor or homolog thereof andcomprises at least one neuropeptide activity.
 36. The vector of claim 35wherein said vector is an expression vector that directs expression ofsaid polynucleotide sequence encoding said neuropeptide.
 37. The vectorof claim 35 wherein said polynucleotide sequence encodes a polypeptidecomprising a polypeptide sequence selected from the group consisting ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 38. Atransformed cell comprising a vector wherein said vector comprises apolynucleotide sequence encoding a polypeptide comprising a GDNFprecursor or homolog thereof, wherein said polypeptide comprises atleast one neuropeptide activity.
 39. The transformed cell of claim 38wherein said vector is an expression vector that directs expression ofsaid polynucleotide sequence encoding said polypeptide.
 40. Thetransformed cell of claim 38 wherein said polynucleotide sequenceencodes a polypeptide comprising a polypeptide sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQID NO:8.
 41. A transformed cell comprising a vector wherein said vectorcomprises a polynucleotide sequence encoding a polypeptide comprising afragment of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 42. Thetransformed cell of claim 41 wherein said vector is an expression vectorthat directs expression of said polynucleotide sequence encoding saidpolypeptide.
 43. The transformed cell of claim 41 wherein saidpolynucleotide sequence encodes a polypeptide comprising a polypeptidesequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 44. A method of producing a GDNFprecursor comprising introducing an expression vector into a cellwherein said expression vector comprises a polynucleotide sequenceencoding a GDNF precursor or homolog thereof, wherein said GDNFprecursor comprises at least one neuropeptide activity.
 45. A method ofproducing a fragment of a GDNF precursor comprising introducing anexpression vector into a cell wherein said expression vector comprises apolynucleotide sequence encoding a fragment of a GDNF precursor orhomolog thereof, wherein said fragment comprises at least oneneuropeptide activity.
 46. A composition comprising at least onepolypeptide and at least one pharmaceutically acceptable carrier,wherein said polypeptide comprises a polypeptide sequence of a GDNFprecursor or homolog thereof, and wherein said polypeptide comprises atleast one neuropeptide activity.
 47. The composition of claim 46 whereinsaid polypeptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8.
 48. A composition comprising at least one polypeptide and at leastone pharmaceutically acceptable carrier, wherein said polypeptidecomprises a fragment of a GDNF precursor or homolog thereof, and whereinsaid polypeptide comprises at least one neuropeptide activity.
 49. Thecomposition of claim 48 wherein said polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 50. A composition comprising anantibody, or portion thereof and at least one pharmaceuticallyacceptable carrier, wherein said antibody or portion thereofspecifically binds to a polypeptide having an amino acid sequence of aGDNF precursor or homolog thereof.
 51. The composition of claim 50wherein said polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQID NO:8.
 52. A composition comprising an antibody or portion thereof andat least one pharmaceutically acceptable carrier, wherein said antibodyof portion thereof specifically binds to a polypeptide having an aminoacid sequence of a fragment of a GDNF precursor or homolog thereof. 53.The composition of claim 52 wherein said polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 54. A method of modulating atleast one activity of a neuron comprising administering to said neuron acomposition comprising a polypeptide wherein said polypeptide comprisesan amino acid sequence of a GDNF precursor or homolog thereof, whereinsaid polypeptide comprises at least one neuropeptide activity.
 55. Themethod of claim 54 wherein said polypeptide is selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.56. A method of modulating at least one activity of a neuron comprisingcontacting with said neuron a composition comprising a polypeptidewherein said polypeptide comprises an amino acid sequence of a fragmentof a GDNF precursor or homolog thereof, wherein said polypeptidecomprises at least one neuropeptide activity.
 57. The method of claim 56wherein said polypeptide is selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 58. (canceled) 59.(canceled)
 60. A method of modulating cRet activity comprisingadministering to a patient in need thereof a composition comprising atleast one polypeptide having an amino acid sequence of a GDNF precursoror homolog thereof, wherein said polypeptide comprises at least oneneuropeptide activity.
 61. A method of modulating cRet activitycomprising administering to a patient in need thereof a compositioncomprising at least one polypeptide having an amino acid sequence of afragment of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 62. The methodof claim 60 or 61 wherein said cRet activity is down-modulated orup-regulated.
 63. A method of modulating NPY—Y_(i) activity comprisingadministering to a patient in need thereof a composition comprising atleast one polypeptide having an amino acid sequence of a GDNF precursoror homolog thereof, wherein said polypeptide comprises at least oneneuropeptide activity.
 64. A method of modulating NPY—Y_(i) activitycomprising administering to a patient in need thereof a compositioncomprising at least one polypeptide having an amino acid sequence of afragment of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 65. The methodof claim 63 or 64 wherein said NPY—Y_(i) activity is down-modulated orup-regulated.
 66. A method of treating a motor disease comprisingadministering to a patient manifesting at least one sign of a motordisease a composition comprising at least one polypeptide having anamino acid sequence of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 67. The methodof claim 66 wherein said polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8.
 68. A method of treating a motor diseasecomprising administering to a patient manifesting at least one sign of amotor disease a composition comprising at least one polypeptide havingan amino acid sequence of a fragment of a GDNF precursor or homologthereof, wherein said polypeptide comprises at least one neuropeptideactivity.
 69. The method of claim 68 wherein said polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 70. The method ofclaim 66 or 68 wherein said motor disease is selected from the groupconsisting of chronic inflammatory demyelinating polyneuropathy,Parkinson's Disease, demyelinating Guillain-Barre syndrome, amyotrophiclateral sclerosis, and motor neuropathy.
 71. A method of treatingneuropathic pain comprising administering to a patient manifesting atleast one sign of a neuropathic pain a composition comprising at leastone polypeptide having an amino acid sequence of a GDNF precursor orhomolog thereof, wherein said polypeptide comprises at least oneneuropeptide activity.
 72. The method of claim 71 wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 73.A method of treating neuropathic pain comprising administering to apatient manifesting at least one sign of a neuropathic pain acomposition comprising at least one polypeptide having an amino acidsequence of a fragment of a GDNF precursor or homolog thereof, whereinsaid polypeptide comprises at least one neuropeptide activity.
 74. Themethod of claim 73 wherein said polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17.
 75. A method of treating a motordisease comprising administering to a patient manifesting at least onesign of a motor disease a composition comprising an antibody or portionthereof that specifically binds to polypeptide having an amino acidsequence of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 76. The methodof claim 75 wherein said polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8.
 77. A method of treating a motor diseasecomprising administering to a patient manifesting at least one sign of amotor disease a composition comprising an antibody or a portion thereofspecifically binds to polypeptide having an amino acid sequence of afragment of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 78. The methodof claim 77 wherein said polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17.
 79. The method of claim 75 or 77 wherein saidmotor disease is selected from the group consisting of chronicinflammatory demyelinating polyneuropathy, Parkinson's Disease,demyelinating Guillain-Barre syndrome, amyotrophic lateral sclerosis,and motor neuropathy.
 80. A method of treating neuropathic paincomprising administering to a patient manifesting at least one sign of aneuropathic pain a composition comprising an antibody or a portionthereof that specifically binds to a polypeptide sequence of a GDNFprecursor or homolog thereof, wherein said polypeptide comprises atleast one neuropeptide activity.
 81. The method of claim 80 wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.82. A method of treating neuropathic pain comprising administering to apatient manifesting at least one sign of a neuropathic pain acomposition comprising an antibody or a portion thereof thatspecifically binds to a polypeptide sequence of a fragment of a GDNFprecursor or homolog thereof, wherein said polypeptide comprises atleast one neuropeptide activity.
 83. The method of claim 82 wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.84. (canceled)
 85. (canceled)
 86. (canceled)
 87. (canceled)
 88. A methodof treating a testicular disorder comprising administering to a patientmanifesting at least one sign of reduced spermatogenesis a compositioncomprising at least one polypeptide having an amino acid sequence of afragment of a GDNF precursor or homolog thereof, wherein saidpolypeptide comprises at least one neuropeptide activity.
 89. The methodof claim 88 wherein said polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17.
 90. A method of modulating excitatoryneurotransmission comprising administering to a subject an effectiveamount of a polypeptide comprising an amino acid sequence of a fragmentof a GDNF precursor or homolog thereof, wherein said polypeptidecomprises at least one neuropeptide activity.
 91. The method of claim 90wherein said polypeptide is selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
 92. The method ofclaim 90 wherein said polypeptide is selected from the group consistingof SEQ ID NO:9, SEQ ID NO:10, and mixtures thereof.
 93. A method oftreating a disorder associated with excitatory neurotransmissioncomprising administering to a subject in need of treatment an effectiveamount of at least one polypeptide comprising a fragment of a GDNFprecursor or homolog thereof, wherein said polypeptide comprises atleast one neuropeptide activity.
 94. The method of claim 93 wherein saiddisorder is selected from the group consisting of epilepsy, stroke,neurogenerative disorders, infantile brain damage, and neuropathic pain.